presentation title arial 28pt bold agilent blue hw tips.pdf · lozano, steve marchand,sebastien...
TRANSCRIPT
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RapidFire
high-throughput
mass spectrometry system
Users Meeting
System Tips and Tricks
Arrin Katz, Sr. Manager, Engineering
Waldbronn, Germany
November 14, 2012
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Confidential
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Outline
RapidFire install base and support worldwide
Mass Spec and Rapid Fire success tips
Setup of a successful RapidFire run
Maintenance for a robust RapidFire system
Troubleshooting and hardware repairs
Advice/tips for multiple systems / users labs
Saving and re-using large batches
Standard and custom cartridges
Additional valve states
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Systems
Close to 80 units in the field
200/300/360
Geography
N. America 60%
Europe 25%
Asia 15%
Mass spectrometers
QQQ 80%
QTOF 20%
RapidFire install base
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10 support engineers
ALEXIS,GERALD
BAUDAILLIER,JOE
CATBAGAN, ALAN
HARTZ, MIKE
LOZANO, STEVE
MARCHAND,SEBASTIEN
SANFORD,HOWARD
STROMAN,MAXCY
THOMSON,REBECCA
TYROS,GINA
6 support engineers
GARCIA,JUAN
LENZ,CHRISTOPH
OBERMAIER,THORSTEN
GOUJON,XAVIER
ROACH,JONATHAN
WAGNER, MORITZ
3 support engineers
QI,YI-ZHOU
WU,HAO
ZHANG,YAO-ZY
4 support engineers
KANAZAWA,YOSHINORI
KUSAKABE,TOMOKAZU
TAKAHASHI,YUTO
TAKATA,YUICHIRO
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blue: I&F trained
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RapidFire/MS System
Sample
5
QQQ
TOF
Agilent Confidential
December 11, 2012
RF ControlPanel
RF User Interface RF Communicator
Data
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MS success tips
• Routine MS maintenance:
– Preventative maintenance
– Mass Calibration
– Cleanliness (source, Q0, Q1…)
• Proper warm up
– P3 at the assay flow rate
– QQQ: 5 minutes
– QTOF: 30 minutes
• Use high purity LC/MS grade solvents
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RF success tips
• Fresh-made buffers
– Weekly (assay dependent; strongly recommend daily)
• Equilibrate the cartridges with blank injections before use
• Keep cartridge logs
– Injection counts of cartridges
– Pressure profiles
– Matrix types (neat solution, organic solution, urine, plasma….)
• Keep Instrument logs
– PM schedule (every six months)
– Repair and troubleshooting history
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Confidentiality LabelA
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Setup of a successful RapidFire run
Check vacuum presence, solvent bottles and waste flasks
Connect to the software (RF Control Panel, UI, Communicator)
Home the stages and go through the sipper configuration wizard
Check column changer is homed
Load cartridges, Go To cartridge of choice
Check valves are homed
Toggle valves (noiselessly) between load and inject
Turn pumps on and off
Verify absence of leaks or overpressures
Refer to pressure history graph
Flush Now
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Clog prevention, identification, and removal
• Sample preparation
– Spin the plate before injection (within 2 hours)
– For overnight runs, transfer supernatant after centrifuging
• Routine flushes (with aqueous solution)
• Extra flush after long sequences with organic solutions
– Move blue PEEK from pump 1 to pump 3
– Valves 1 at inject position and valve 4 at load position
– Turn on pump 3 at 8 mL/min
• Flush with both aqueous and organic solutions if a minor clog occurs
• Aspiration verification (25-40 sec/mL)
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Troubleshooting: Aspiration issues
• vacuum connections
- 70 kPa
• Sip Height deep enough?
• aspiration valves in inject,
through sipper 1 mL water in 25 - 40 s
through loop significantly faster?
• flush in both directions
with both solvents
• sip sensor calibration (“ – “ button)
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• Sapphire window (transparent, chemically inert)
• Fibers: one emits, one receives light
• bright / liquid ~ 2 X dark / air readout
• Calibration using the static “–“ button
- tube filled with water
- press “ - ” for 3 seconds to enter static teach mode
- when “ 1st ” flashes, present air, then press “ - ” briefly
- when “ 2nd ” flashes, present water, then press “ - ”
- “ pass ” will flash with contrast grade displayed (e.g. 2000)
- check with alternating full / empty wells
Troubleshooting: Sip sensor calibration
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Troubleshooting: Home a valve
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Troubleshooting: Home a valve
• Under System Tools\ Valve Tuner, press Find
with the Limited Homing box checked (60o only)
Tip: first press the E-STOP and manually
rotate the coupling counterclockwise
• Make sure load > 50 (positive)
inject < - 50 (negative)
Home 0 must be between inject and load
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Troubleshooting: Overpressure
• Determine pump that overpressured
• Check pressure as you toggle valves from
load to inject
• Replace cartridge
• Trace fluidic lines and monitor pressure as
you connect/disconnect
• Replace tubing
• Flush groove in valve
• Replace valve
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Troubleshooting:
Change a valve • disconnect tubing
loosen coupling’s bottom screw
remove (9/64 hex-key) top two screws
• V1 and V3 outward-facing sticker, V2 inward
• slide in new valve (with adapters, if easier)
tighten two screws
reconnect tubing
• Home valve
tighten coupling’s bottom screw
define valve’s load and inject positions
by rotating coupling manually
• save new configuration values
by clicking Set (underneath Cur.)
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Troubleshooting: Initialize NI boards
• Go to Start > All Programs > National Instruments > Measurement & Automation
• The Devices and Interfaces tree expands to - PCI-x (1) “linear motions”
- PCI-x (2) “rotary motions”
• Highlight each board - Reset Device
- Initialize
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• Keep a supply of spare parts in house:
• Nano-valve *** new part # G9203-80026
• RF specific fittings
• RF specific tubing
• Needles
• Cartridges
• Consult us for recommended parts and
quantities
Spare parts – minimize downtime
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RapidFire experiment building blocks
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Batch related definitions
Batch file: a .RFBAT recipe enumerating how to run one or multiple plates of a RapidFire‐
MassHunter experiment by specifying for each plate which (a) platemap to follow, and for each
sequence within each plate which (b) .RFCFG RapidFire Method, (c) solid‐phase extraction (SPE)
cartridge and (d) .M MS Method to use.
MS Method: a .M data acquisition method in MassHunter, saved in D:\ MassHunter\ Methods\
RapidFire\.
Platemap file: a .RFMAP profile of the series of sequences, and of wells within each sequence, to be
visited during the analytical read of a 96‐ or 384‐well microtiter plate during a RapidFire‐MassHunter
experiment.
RapidFire Method: a .RFCFG summary of RapidFire parameters to apply to a given sequence,
including (a) RapidFire cycle timing, (b) pump flow rates, (c) sip height, (d) occurrence of “wash
between sips” (dips of the sipper tube and its guiding needle in both wash chimneys in‐between
injections), and (e) existence of blank injections (full cycles in the organic wash chimney in‐between
sample injections).
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Multiple plates / users / mass spectrometers
Tips for lab sanity
Plate types:
• Single cfgs directory C:\ Program Files\ Agilent\ RapidFire\ cfgs
• Multiple .PLATECFG configuration files to save all parameters for all plates
- teach Twister vertical adjustment for stage and hotels
- Sipper Configuration Wizard for each plate type
Users
• Users can save batch files in separate, project-specific directories
Mass spectrometers
• Same IP address for all MS = 192.168.254.1
RF = 192.168.254.2
• RF Communicator now stops 2 .EXE processes first
RF Stop Processes new icon too
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Prepare and re-use large batches
• In Sequences mode, with barcodes as Plate Names, an exhaustive set of recipes
(= platemaps + .RFCFG methods + SPE cartridges + .M or .DAM methods)
can be saved.
• As the batch is run, only the plates with “today’s barcodes” will be read.
Other lines in the batch will be ignored, they won’t hurt or delay the run!
Methods
now linked
to MS
method dir.
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Selection chart for RapidFire SPE cartridges
Type Packing Typical Applications Part #
A C4 Small molecules, peptides, oligos G9203A
A2 C4 Small molecules, peptides, oligos G9525A
B Cyano Hydrophobic compounds G9204A
C C18 Proteins G9205A
D Hypercarb Hydrophilic compounds, small molecules G9206A
E C8 Proteins G9207A
F Phenyl Aromatic compounds G9208A
H HILIC Hydrophilic compounds, small molecules G9209A
O Blank Testing G9211A
Z Custom Custom applications G9210A
Inlet
Frit
Frit
Outlet
SPE
Packing
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• Custom packed per customer specifications
• Order submitted to Agilent (minimum quantity 5, in 5 unit increments)
• Packing material shipped to Agilent Wakefield
• 45 days lead time
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Custom Cartridges – G9210A
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Matrix bottles – RF300/360
Four matrix bottles: 10 mL/vial
• External standards
• QC samples
• Special wash solutions
• Calibration curve
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P/N 5190-5799 P/N 5188-9057
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Plate Seals
• Agilent PlateLoc Thermal Microplate Sealer P/N G5402A
• Agilent peelable aluminum seal P/N 24210-001
• Agilent pierceable aluminum seal P/N 06644-001
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• Find local support contact info at
http://www.chem.agilent.com/en-US/Contact-US/Pages/ContactUs.aspx
• Open service request (under warranty or service agreement)
– serial number, e.g. RF-MS XX (up to 57), USXXXXXXXX, or
SGXXXXXXXX
(on the instrument frame)
• Request quote for onsite services (no warranty or service contract)
• Request quote for parts and consumables
• Keep record of the service request number (SR#)
• Save log files and screenshot s of error messages
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Service contact information
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User feedback and wish list
• Reduce min. volume required
• Increase unattended operation time – over weekend
• Sample prep options
• Reduce volume for matrix stations
• Temperature control
• Interface with existing lab robotics
• Provide test data for cartridges (performance variation)
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Questions and discussion
What improvements would you like to see?
What tips do you have for successful operation?
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Agilent RapidFire High-
throughput Mass
Spectrometry Software
Summary of Features
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Powerful Tools - Simple Workflow
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Minimal
Operator
Intervention
Flexible
Sample
Information
Real-time
run
monitoring
Powerful
Quantitative
Analysis
Customizable
Report
Generation
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Flexible Sample Information Format
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Import sample
info from LIMS
Multiple user defined
columns available
Add method to input
file for minimal
operator intervention
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Flexible Sample Information Format
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Converter allows
you to use your
own column
headers in sample
input
Set up conversion to MassHunter field headers once and
then all experiments will be processed automatically
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Run Template
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Run Template = a one plate batch defining sequences, RF method, cartridge and MS method
Run Template
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Minimal Operator Intervention
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1. Create run templates in advance
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Minimal Operator Intervention
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2. Load Sample File
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Minimal Operator Intervention
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3. Press Start
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Real-time Run Monitoring
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RapidFire Injections
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Real-time Run Monitoring
December 11, 2012
Agilent Confidential
38
Monitor the TIC in real-time with MassHunter Acquisition software
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MassHunter Qual
December 11, 2012
Agilent Confidential
39
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MassHunter Quant
December 11, 2012
Agilent Confidential
40
Load .d files containing all sample info,
run parameters and mass spec data
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Powerful Quantitative Analysis
December 11, 2012
Agilent Confidential
41
Complete MassHunter Quantitative Workstation features
available on request
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Customizable Report Generation
December 11, 2012
Agilent Confidential
42
Excel PDF